Lignocellulosic biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful products. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol and other products. In order to convert these fractions, the cellulose and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; this hydrolysis has historically proven to be problematic.
Biologically mediated processes are promising avenues for the conversion of lignocellulosic biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two basic strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.
Three major types of enzymatic activities are required for native cellulose degradation: The first type are endoglucanases (1,4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (1,4-O-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. The third type are β-glucosidases (β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units.
Bakers' yeast (Saccharomyces cerevisiae) remains the preferred micro-organism for the production of ethanol (Hahn-Hägerdal, B., et al., Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Favorable attributes of this microbe include (i) high productivity at close to theoretical yields (0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making, and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolyzates resulting from biomass pretreatment.
One major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins. In attempt to address this problem, several heterologous cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the degradation of cellulosic derivatives (Van Rensburg, P., et al., Yeast 14:67-76 (1998)), or growth on cellobiose (Van Rooyen, R., et al., J. Biotech. 120:284-295 (2005); McBride, J. E., et al., Enzyme Microb. Techol. 37:93-101 (2005)). However current levels of expression and specific activity of cellulases heterologously expressed in yeast are still not sufficient to enable efficient growth and ethanol production by yeast on cellulosic substrates without externally added enzymes. There remains a significant need for improvement in the amount of cellulase activity in order to attain the goal of achieving a consolidated bioprocessing (CBP) system capable of efficiently and cost-effectively converting cellulosic substrates to ethanol or other useful products.
Heterologous cellulase enzymes are usually produced by recombinant organisms in such low concentrations that the amount of saccharified substrate available is unable to sustain growth of the organisms. Cellulase enzymes can be expressed as secreted enzymes that are not purposely attached to the yeast cell wall, resulting in a physical separation of the cellulase enzyme and the cell that made it, or they can be expressed tethered to the cell surface. This covalent linkage to the cell surface may provide benefits due to the ability to select enhanced cellulase secreting organisms in liquid culture, and/or because of the concentration increase of cellulase close to a particular cell. However, tethered cellulase expression suffers from a limited surface area on the cell surface to bind to, and it is not clear whether secreting or tethering cellulase enzymes will ultimately provide better results.
Various cellulase genes have been expressed in Saccharomyces cerevisiae and other yeasts with the aim of direct ethanol production from cellulose, including components of both non-complexed and complexed cellulase systems (see comprehensive review in (Gal L., et al., J. Bacteriol. 179(21):6595-601 (1997); van Zyl W. H., et al., Adv. Biochem. Eng. Biotechnol. 108:205-35 (2007)). In one such attempt, a rudimentary non-complexed cellulase system consisting of a single endoglucanase and an single beta-glucosidase allowed the yeast to convert phosphoric acid swollen cellulose (PASC) directly to ethanol (Den Haan R., Metab. Eng. 9(1):87-94 (2007)).
Complexed cellulases, or cellulosomes (first described by (Lamed R., et al., J. Bacteriol. 156(2):828-36 (1983)), on the other hand, are multi-protein complexes comprised of catalytic component linked via binding domains called “dockerins” to a structural component called a “scaffoldin.” This structural protein, which may or may not contain a catalytic domain, often contains a cellulose binding module, in addition to domains called “cohesins,” which serve to bind to the dockerins found on the catalytic components. The catalytic components can include cellulases with similar activities to those found in non-complexed cellulase systems, and can also include a wide range of hydrolyzing activities, such as hemicellulase and pectinase activities.
The activity of non-complexed and complexed cellulase systems has rarely been directly compared on a consistent basis. However, specific activity data collected broadly from across the literature indicate that cellulosomes are substantially (˜5 to 10 times) more active on a mass basis than non-complexed systems (Lynd L., et al., Microbiol. Mol. Biol. Rev. 66:506 (2002)). Additionally, it is well-established that organisms with cellulosomes, like C. thermocellum, can grow at relatively high rates on crystalline cellulose, including pretreated lignocellulose (Lynd L., et al., Microbiol. Mol. Biol. Rev. 66:506 (2002)). Cellulosomes have been found mainly in anaerobic environments, and largely in bacterial species. However, species of anaerobic fungi that live in the rumen have also been shown to have cellulosomes, with very high cellulase specific activity (Wilson C. A. and Wood T. M., Appl. Microbiol. Biotechnol. 37(1):125-9 (1992)).
However, organisms that contain cellulosomes lack the ability to form useful products, such as ethanol, in appreciable quantities. Therefore, there is a need in the art to generate organisms which benefit from the increased cellulolytic capacity of cellulosomes while also having the ability to convert the liberated sugars to useful products, such as ethanol.
Knowledge of complexed cellulase expression in yeast is rudimentary. Production of a scaffoldin in yeast has been accomplished, but simultaneous expression of other necessary components of a cellulosome has not been demonstrated. Additionally, no cellulosome reconstruction has been shown to allow the direct conversion of cellulose to ethanol or other useful products. Constructing cellulosomes in yeast for CBP has a great deal of potential because of the high specific activity of cellulosomes might lead to more efficient production of useful products.
Because heterologous cellulase enzymes are often poorly expressed and secreted by yeast and, because they are the rate limiting factor for cellulose hydrolysis, they need to be expressed as highly as possible. Relative to non-complexed cellulases, as little as a fifth to a tenth of the expression level might be required to achieve similar cellulose hydrolysis rates.
The present invention provides for the heterologous expression of cellulosomes in various microbes as well as methods for their use.